Influenza NS1-Mediated N(6)-Methyladenosine Modification Controls NS mRNA Splicing and Nuclear Exportation

Abstract

<p>N⁶-methyladenosine (m6A) modification is a common RNA modification that has been found in influenza virus (IAV) RNAs since 1976, which has a profound impact on viral replication and pathogenicity. A recent study mapped the m6A sites on the IAV-PR8 strain using PAR-CLIP, in which we found two peaks in the spliced gene--NS segment. Since m6A modification has been proposed to regulate mRNA splicing and nuclear export, we would like to investigate the role of m6A in the splicing and nuclear exportation of the NS segment. Here, we confirmed that m6A addition indeed regulated NS segment splicing and nuclear exportation and demonstrated that this effect was largely mediated by the viral protein NS1 and nuclear m6A reader YTHDC1. To address the role of m6A, we constructed an m6A-deficient IAV mutant. As a result, the NS mRNA aggregated in the nucleus, and spliced form of the NS segment increased during infection with the mutant. Both in vitro and in vivo assays confirmed that NS1 protein inhibited the addition of m6A modification. In addition, we showed that knocking out YTHDC1 protein increased the level of unspliced NS segment RNA. YTHDC1 accomplished this inhibition by blocking the binding of a splicing factor called SRSF3 to the NS mRNA. Consequently, the decreased binding of SRSF3 resulted in a reduced production of the spliced form of NS segment. Our findings provided the direct evidence that NS1 protein of IAV regulated its own splicing by controlling the addition of m6A modification and recruiting m6A reader YTHDC1. This recruitment of YTHDC1 blocks the access of the splicing factor SRSF3 to the NS mRNA, thereby influencing the splicing process and affecting viral replication and pathogenicity.<br></p>